ABSTRACT/PROJECT SUMMARY It remains elusive how the human fungal pathogen Aspergillus fumigatus is able to adapt to and persevere within the hypoxic microenvironment of the host, a characteristic critical to pathogenesis and disease progression. The goal of this proposal is to expand our understanding of fungal pathogenesis, focusing on the novel role of fungal collagen-like proteins and their influence on the fungal extracellular matrix and hypoxia fitness. The A. fumigatus collagen-like protein, CgnA, was identified for its critical role in the hypoxia fitness of an in vitro hypoxia-evolved strain, where a null mutant of cgnA in this background suppresses the in vitro hypoxic growth and the altered colony morphology of the hypoxia-evolved strain. Furthermore, the hypoxia-evolved strain displays a complete detachment of the fungal extracellular matrix (ECM) component galactosaminogalactan (GAG), revealing bare hyphae, a phenotype repeated in the parental strain with over expression of the CgnA upstream regulator. Given the well-characterized role of GAG in the virulence of A. fumigatus, expression of CgnA is likely to significantly alter the host response to A. fumigatus in vivo. However, the role of GAG in hypoxia adaptation, and the influence of secreted GAG versus attached GAG within the host microenvironment remain unexplored and are the focus of this proposal. To address these novel roles for CgnA and the extracellular matrix, electron microscopy and adherence assays with exogenous GAG will be performed. To determine the role of CgnA in hypoxia fitness and fungal pathogenesis, a nanoparticle oxygen reporter will be used to quantify hypoxic stress in the presence and absence of GAG, and fungal host survival and in vivo immunological assays will be performed, respectively. The results of this proposal will define a novel link between unstudied collagen-like fungal proteins, the extracellular matrix, hypoxia fitness, and fungal virulence, an area that has not been previously explored in human pathogenic fungi.